Highly efficient preservation techniques for biological cells or tissues are desired in various fields of industry. For example, in bovine embryo transfer technology, embryos need to be transferred in consideration of the estrus cycle of a recipient cow, and to this end, embryos cryopreserved in advance are thawed and transferred during a suitable phase of the estrus cycle. In human fertility treatment, eggs or ovaries harvested from a woman's body are cryopreserved until an appropriate timing for implantation, and the cryopreserved eggs are thawed before use in implantation.
In general, cells or tissues harvested from living bodies gradually become inactive even when they are in culture medium, and hence their long-term in vitro culture is undesirable. For this reason, techniques for long-term preservation of biological samples without the loss of biological activity are essential. Highly efficient preservation techniques would enable more accurate analysis of cells or tissues harvested from living bodies. Such preservation techniques would enable implantation of biological samples with their biological activity kept at a higher level, thus resulting in an improvement in the engraftment rate. It would be also possible that artificial tissues for implantation such as in vitro cultured skin or so-called cell sheet formed in vitro are produced in advance and stored until needed. Therefore, highly efficient preservation techniques are widely demanded in industry as well as in medical science.
One of the known methods for preserving cells or tissues is slow freezing, for example. In this method, cells or tissues are immersed in a preservation solution prepared by adding a cryoprotectant to a physiological solution such as phosphate buffered saline. Compounds used as the cryoprotectant include glycerol and ethylene glycol. The cells or tissues immersed in the preservation solution are cooled down to −30 to −35° C. at a relatively slow cooling rate (for example, 0.3 to 0.5° C./min.), and thereby the fluid inside the cells or tissues and the solution outside the cells or tissues are sufficiently cooled and become viscous. Further cooling down the cells or tissues in such a state in the preservation solution to the temperature of liquid nitrogen (−196° C.) allows both the inside of the cells or tissues and the closely surrounding solution to become amorphous solid, that is, vitrify. The vitrification (that is, solidification) inside and outside the cells or tissues arrests substantial molecular movement. Thus, the vitrified cells or tissues can be semipermanently preserved in liquid nitrogen.
However, the slow freezing method requires relatively slow-rate cooling, and thus prolongs the process of cryopreservation. In addition, this method disadvantageously needs the use of a temperature controlling instrument or device. Furthermore, the slow freezing method cannot avoid ice crystal formation in the preservation solution outside the cells or tissues, which can cause physical damage to the cells or tissues.
A proposed solution to the problems of the slow freezing method is vitrification-based preservation. The vitrification-based preservation uses a principle that the addition of a large amount of a cryoprotectant, such as glycerol, ethylene glycol and DMSO (dimethyl sulfoxide), to water decreases the freezing point of water, thus preventing ice crystal formation at sub-zero temperatures. Such a cryoprotectant-containing aqueous solution solidifies without ice crystal formation when rapidly cooled in liquid nitrogen. This solidification is called vitrification freezing. The aqueous solution containing a large amount of a cryoprotectant is called a vitrification solution.
The specific procedure of the vitrification-based preservation is to immerse cells or tissues in a vitrification solution and to cool them at the temperature of liquid nitrogen (−196° C.). Such a simple and quick process of cryopreservation can be completed in less time without the use of any temperature controlling instrument or device.
The vitrification-based preservation does not cause ice crystal formation either inside or outside the cells, and thus can prevent physical damage (freezing damage) to the cells at the time of freezing and thawing. However, the high concentration of the cryoprotectant contained in the vitrification solution is chemically toxic, and therefore, the volume of the vitrification solution used in the cryopreservation process is preferably not more than necessary. In addition, the duration of exposure of the cells or tissues to the vitrification solution, that is, the time to freezing is preferably short. Furthermore, the vitrification solution should be diluted quickly and immediately after thawing.
The vitrification-based cryopreservation of cells or tissues as described above has been reported, and various examples using different methods and different kinds of cells and tissues have been presented. For example, Patent Literature 1 describes that the application of vitrification-based preservation to reproductive or somatic cells of animal or human origin is very useful in terms of the cell viability after cryopreservation and thawing.
The vitrification-based preservation is a technique which has been developed mainly using human reproductive cells, and more recently, its application to iPS or ES cells has been widely examined. Non Patent Literature 1 describes the effectiveness of vitrification-based preservation of Drosophila embryos. Patent Literature 2 and Non Patent Literature 2 describe the effectiveness of vitrification-based preservation of plant culture cells and tissues. As just described, vitrification-based preservation is known to be useful for a wide range and different kinds of cells and tissues.
The devices and procedures for more efficient vitrification-based preservation have been reported in publications. For example, Patent Literature 3 reports an attempt to improve the recovery rate of eggs or embryos cryopreserved by vitrification in a straw. The procedure includes vitrifying and preserving eggs or embryos in a claimed straw filled with a vitrification solution, and bringing the cryopreserved eggs or embryos into contact with a diluent in the straw quickly and immediately after thawing.
Patent Literature 4 proposes a cryopreservation method comprising placing eggs or embryos with a vitrification solution on a vitrification solution-removing material and removing excess vitrification solution surrounding the eggs or embryos by downward suction through the vitrification solution-removing material. The eggs or embryos cryopreserved by this method are shown to retain high viability. Examples of the vitrification solution-removing material described in the literature include wire mesh and perforated films made of natural substances, such as paper, or synthetic resins.
Patent Literature 5 proposes a cryopreservation method comprising absorbing excess vitrification solution surrounding eggs or embryos with an absorber such as a filter paper. The eggs or embryos cryopreserved by this method are shown to retain high viability.
Patent Literature 6 and Patent Literature 7 propose the so-called Cryotop method, which is used in the field of fertility treatment in humans. This cryopreservation method uses a flexible, clear and colorless film strip as an egg holding strip and comprises depositing eggs or embryos with a very small amount of a vitrification solution on the film under a microscope.